Use of cysteamine and derivatives thereof to suppress tumor metastases

ABSTRACT

The present Disclosure is directed to methods for inhibiting or suppressing metastasis of a tumor in a mammalian subject using a cysteamine product, e.g., cysteamine or cystamine or a derivative thereof. Also described herein is a method for treating pancreatic cancer in a mammalian subject by administering a cysteamine product described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/699,745 filed on Sep. 8, 2017, which is a continuation of U.S.application Ser. No. 15/065,355 filed on Mar. 9, 2016, which is acontinuation of U.S. application Ser. No. 14/256,687 filed on Apr. 18,2014, which claims the priority benefit of U.S. Provisional PatentApplication No. 61/814,010, filed Apr. 19, 2013, the disclosures ofwhich are incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was funded by the National Institutes of Health of theUnited States of America. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to the use of cysteamine or cystamine orderivatives thereof to treat, inhibit or suppress metastasis of a tumor.The present invention also related to the use of cysteamine or cystamineor derivatives thereof to treat pancreatic cancer.

BACKGROUND

Cysteamine (HS—CH₂—CH₂—NH₂) is a small sulfhydryl compound that is ableto cross cell membranes easily due to its small size. Cysteamine plays arole in formation of the protein glutathione (GSH) precursor, and iscurrently FDA approved for use in the treatment of cystinosis, anintra-lysosomal cystine storage disorder. In cystinosis, cysteamine actsby converting cystine to cysteine and cysteine-cysteamine mixeddisulfide, which are then both able to leave the lysosome through thecysteine and lysine transporters respectively (Gahl et al., N Engl J Med2002; 347(2):111-21). Within the cytosol the mixed disulfide can bereduced by its reaction with glutathione and the cysteine released canbe used for further GSH synthesis. Treatment with cysteamine has beenshown to result in lowering of intracellular cystine levels incirculating leukocytes (Dohil et al., J. Pediatr 148(6):764-9, 2006).

Cysteamine is also discussed in (Prescott et al., Lancet 1972;2(7778):652; Prescott et al., Br Med J 1978; 1(6116):856-7; Mitchell etal., Clin Pharmacol Ther 1974; 16(4):676-84; Toxicol Appl Pharmacol.1979 48(2):221-8; Qiu et al., World J Gastroenterol. 13:4328-32, 2007.Unfortunately, the sustained concentrations of cysteamine necessary fortherapeutic effect are difficult to maintain due to rapid metabolism andclearance of cysteamine from the body, with nearly all administeredcysteamine converted to taurine in a matter of hours. These difficultiesare transferred to patients in the form of high dosing levels andfrequencies, with all of the consequent unpleasant side effectsassociated with cysteamine (e.g., gastrointestinal distress and bodyodor) See the package insert for CYSTAGON® (cysteamine bitartrate).International Publication No. WO 2007/079670 discloses entericallycoated cysteamine products and a method of reducing dosing frequency ofcysteamine.

Cysteamine is addressed in International Patent Application Nos. WO2009/070781, and WO 2007/089670, and U.S. Patent Publication Nos.20110070272, 20090048154, and 20050245433.

In the cancer field, studies have reported anti-cancer effects ofcysteamine with respect to cancer development and proliferation.Cysteamine prevented the development of metaplasia and carcinogenesis ofmammary tumor and gastric cancers induced chemically and by radiation(6-8). Cysteamine by itself or conjugated with nanoparticles or othercompounds suppress cancer cell proliferation derived from neuralneoplastic tumors (9), SMIVIC-7721 hepatocellular carcinoma (10), breastcancer (11), and melanoma cell lines (12) in vitro.

SUMMARY

In one aspect, described herein is a method of inhibiting or suppressingmetastasis of a tumor in a mammalian subject comprising administeringcysteamine, cystamine or pharmaceutically acceptable salts thereof tothe subject in an amount effective to inhibit metastasis of the tumor.

In another aspect, described herein is a method of treating pancreaticcancer in a mammalian subject comprising administering cysteamine,cystamine or pharmaceutically acceptable salts thereof to the subject inan amount effective to treat the cancer.

In various embodiments, the cysteamine, cystamine or pharmaceuticallyacceptable salts thereof is administered orally, optionally beingformulated for delayed release. In some embodiments, the cysteamine,cystamine or pharmaceutically acceptable salt thereof is formulated in aenterically coated tablet or capsule.

The delayed release formulation, in some embodiments, comprises anenteric coating that releases the cysteamine or cystamine when theformulation reaches the small intestine or a region of thegastrointestinal tract of a subject in which the pH is greater thanabout pH 4.5.

In some embodiments, the cysteamine, cystamine or pharmaceuticallyacceptable salts thereof is administered less than four times a day(e.g., three times, two times or once a day). In some embodiments, thecysteamine, cystamine or pharmaceutically acceptable salts thereof isadministered twice a day.

In various embodiments of the disclosure, the cysteamine, cystamine orpharmaceutically acceptable salt thereof is administered in a dose ofabout of 0.01 mg to 1000 mg per kg (mg/kg) of body weight per day. Insome embodiments, the cysteamine, cystamine or pharmaceuticallyacceptable salt thereof is administered at a daily dose ranging fromabout 10 mg/kg to about 250 mg/kg, or from about 100 mg/kg to about 250mg/kg, or from about 60 mg/kg to about 100 mg/kg or from about 50 mg/kgto about 90 mg/kg, or from about 30 mg/kg to about 80 mg/kg, or fromabout 20 mg/kg to about 60 mg/kg, or from about 10 mg/kg to about 50mg/kg. Further, the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg,10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg,300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, and may increaseby 25 mg/kg increments up to 1000 mg/kg, or may range between any two ofthe foregoing values. In some embodiments, the cysteamine product isadministered at a total daily dose of from approximately 0.25 g/m² to4.0 g/m² body surface area, about 0.5-2.0 g/m² body surface area, or1-1.5 g/m² body surface area, or 1-1.95 g/m² body surface area, or 0.5-1g/m² body surface area, or about 0.7-0.8 g/m² body surface area, orabout 1.35 g/m² body surface area, or about 1.3 to about 1.95grams/m²/day, or about 0.5 to about 1.5 grams/m2/day, or about 0.5 toabout 1.0 grams/m²/day, e.g., at least about 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², orup to about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.2, 2.5, 2.7, 3.0, 3.25, 3.5 or 3.75 g/m² or may range between anytwo of the foregoing values.

In some embodiments, the administering results in increased thiol levelscompared to levels before administration of the cysteamine, cystamine orpharmaceutically acceptable salts thereof.

In some embodiments, the administering modulates enzymatic activity of amatrix metalloproteinase (MMP, e.g., MMP-1, MMP-2, MMP-3, MMP-4, MMP-5,MMP-6, MMP-6, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 and/orMMP-14.) in a cancer cell of the tumor. In some embodiments, enzymaticactivity of MMP is decreased in a primary tumor.

In some embodiments, the administering decreases metastatic nodulesand/or ascites in the subject.

The methods described herein are useful for the inhibition orsuppression of metastasis of any cancer. In some embodiments, the canceris selected from the group consisting of breast cancer, melanoma,prostate cancer, pancreatic cancer, head and neck cancer, lung cancer,non small-cell lung carcinoma, renal cancer, colorectal cancer, coloncancer, ovarian cancer, liver cancer and gastric cancer.

Aspects of the invention that are described herein as methods(especially methods that involve treatment) can alternatively bedescribed as (medical) uses of cysteamine, cystamine or pharmaceuticallysalts thereof. For example, in one variation, described herein the useof cysteamine, cystamine or pharmaceutically acceptable salts thereof toinhibit or suppress metastasis of a tumor. In another variation,described herein is the use of cysteamine, cystamine or pharmaceuticallyacceptable salts thereof to treat pancreatic cancer.

The agents and compositions described herein for use in treatment arethemselves aspects of the invention also, e.g., as compositions ofmatter.

In the treatment methods (or uses) described herein, the methodsoptionally comprise administering an adjunct cancer therapy to thesubject in combination with the cysteamine, cystamine orpharmaceutically acceptable salts thereof. In some embodiments, theadjunct cancer therapy is selected from the group consisting ofchemotherapy, surgery, radiotherapy, cancer vaccines, immunotherapy,gene therapy, thermotherapy and laser therapy.

In some embodiments, the methods (or uses) described herein furthercomprise administering a further therapeutic agent selected from thegroup consisting of an MMP inhibitor, a chemotherapeutic agent, a growthinhibitory agent and a cytokine.

With respect to any combination treatments described herein, thecysteamine, cystamine or pharmaceutically acceptable salts thereof canbe administered simultaneously with the other active agents, which maybe in admixture with the agent or may be in a separate composition. Eachcomposition preferably includes a pharmaceutically acceptable diluent,adjuvant, or carrier. When the agents are separately administered, theymay be administered in any order.

In another aspect, described herein is a method of decreasing matrixmetalloproteinase (MMP) enzymatic activity in a cancer cell comprisingcontacting the cell with cysteamine, cystamine or pharmaceuticallyacceptable salts thereof in an amount effective to decrease MMPenzymatic activity in the cancer cell. In various embodiments, the MMPis selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-4,MMP-5, MMP-6, MMP-6, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 andMMP-14. In various embodiments, the MMP is selected from the groupconsisting of MMP-9, MMP-12 and MMP-14. In various embodiments, the MMPis MMP-9.

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations defined by specific paragraphs above. For example,certain aspects of the invention that are described as a genus, and itshould be understood that every member of a genus is, individually, anaspect of the invention. Also, aspects described as a genus or selectinga member of a genus, should be understood to embrace combinations of twoor more members of the genus. Although the applicant(s) invented thefull scope of the invention described herein, the applicants do notintend to paragraph subject matter described in the prior art work ofothers. Therefore, in the event that statutory prior art within thescope of a paragraph is brought to the attention of the applicant(s) bya Patent Office or other entity or individual, the applicant(s) reservethe right to exercise amendment rights under applicable patent laws toredefine the subject matter of such a paragraph to specifically excludesuch statutory prior art or obvious variations of statutory prior artfrom the scope of such a paragraph. Variations of the invention definedby such amended paragraphs also are intended as aspects of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B provide Kaplan-Meier survival curves of mice harboringHS766T (FIG. 1A) and MIA-PaCa2 (FIG. 1B) tumors.

DETAILED DESCRIPTION

The present disclosure relates, in general, to the discovery of theanti-invasive and/or anti-metastatic effects of cysteamine in humancancers.

Definitions

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a derivative”includes a plurality of such derivatives and reference to “a patient”includes reference to one or more patients and so forth.

Also, the use of“or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and products, the exemplary methods,devices and materials are described herein.

The documents discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure. Each document is incorporated by reference in itsentirety with particular attention to the disclosure for which it iscited.

The following references provide one of skill with a general definitionof many of the terms used in this disclosure: Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THECAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THEGLOSSARY OF GENETICS, 5TH ED., R. Rieger, et al. (eds.), Springer Verlag(1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY(1991).

As used herein, a “therapeutically effective amount” or “effectiveamount” refers to that amount of a cysteamine product, e.g., cysteamine,cystamine or a pharmaceutically acceptable salt thereof, sufficient toresult in amelioration of symptoms, for example, treatment, healing,prevention or amelioration of the relevant medical condition, or anincrease in rate of treatment, healing, prevention or amelioration ofsuch conditions, typically providing a statistically significantimprovement in the treated patient population. When referencing anindividual active ingredient, administered alone, a therapeuticallyeffective dose refers to that ingredient alone. When referring to acombination, a therapeutically effective dose refers to combined amountsof the active ingredients that result in the therapeutic effect, whetheradministered in combination, including serially or simultaneously. Invarious embodiments, a therapeutically effective amount of thecysteamine product ameliorates symptoms associated with various cancers,including but not limited to, loss of appetite, oral pain, upperabdominal pain, fatigue, abdominal swelling, persistent aches, bonepain, nausea, vomiting, constipation, weight loss, headaches, rectalbleeding, night sweats, digestive discomfort, and painful urination.

“Treatment” refers to prophylactic treatment or therapeutic treatment.In certain embodiments, “treatment” refers to administration of acompound or composition to a subject for therapeutic or prophylacticpurposes.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs or symptoms of pathology for the purpose of diminishingor eliminating those signs or symptoms. The signs or symptoms may bebiochemical, cellular, histological, functional or physical, subjectiveor objective.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease, for the purpose of decreasing the risk of developing pathology.The compounds or compositions of the disclosure may be given as aprophylactic treatment to reduce the likelihood of developing apathology or to minimize the severity of the pathology, if developed.

“Diagnostic” means identifying the presence, extent and/or nature of apathologic condition. Diagnostic methods differ in their specificity andselectivity. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in a subject animal, including humans and mammals. Apharmaceutical composition comprises a therapeutically effective amountof a cysteamine product, optionally another biologically active agent,and optionally a pharmaceutically acceptable excipient, carrier ordiluent. In an embodiment, a pharmaceutical composition encompasses acomposition comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product thatresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present disclosure encompass anycomposition made by admixing a compound of the disclosure and apharmaceutically acceptable excipient, carrier or diluent.

“Pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical carriers, buffers, and the like, such as a phosphatebuffered saline solution, 5% aqueous solution of dextrose, and emulsions(e.g., an oil/water or water/oil emulsion). Non-limiting examples ofexcipients include adjuvants, binders, fillers, diluents, disintegrants,emulsifying agents, wetting agents, lubricants, glidants, sweeteningagents, flavoring agents, and coloring agents. Suitable pharmaceuticalcarriers, excipients and diluents are described in Remington'sPharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).Preferred pharmaceutical carriers depend upon the intended mode ofadministration of the active agent. Typical modes of administrationinclude enteral (e.g., oral) or parenteral (e.g., subcutaneous,intramuscular, intravenous or intraperitoneal injection; or topical,transdermal, or transmucosal administration).

A “pharmaceutically acceptable salt” is a salt that can be formulatedinto a compound for pharmaceutical use, including but not limited tometal salts (e.g., sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines. Examples of cysteamine derivativesinclude hydrochloride, bitartrate and phosphocysteamine derivatives.Cystamine and cystamine derivatives include sulfated cystamine.

As used herein “pharmaceutically acceptable” or “pharmacologicallyacceptable” salt, ester or other derivative of an active agent comprise,for example, salts, esters or other derivatives refers to a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be administered to an individual without causing any undesirablebiological effects or without interacting in a deleterious manner withany of the components of the composition in which it is contained orwith any components present on or in the body of the individual.

As used herein, the term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a compound ofthe disclosure calculated in an amount sufficient to produce the desiredeffect, optionally in association with a pharmaceutically acceptableexcipient, diluent, carrier or vehicle. The specifications for the novelunit dosage forms of the present disclosure depend on the particularcompound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

As used herein, the term “subject” encompasses mammals. Examples ofmammals include, but are not limited to, any member of the mammalianclass: humans, non-human primates such as chimpanzees, and other apesand monkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. The term does not denote a particular age or gender. In variousembodiments the subject is human.

Cancer

The invention is contemplated to be useful with respect to any cancertype. Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, urinarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma,Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular(eye) melanoma, merkel cell carcinoma, mesothelioma malignant,mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer ofthe tongue, multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor. In some embodiments,the tumor is associated with a cancer selected from the group consistingof breast cancer, melanoma, prostate cancer, pancreatic cancer, head andneck cancer, lung cancer, non small-cell lung carcinoma, renal cancer,colorectal cancer, colon cancer, ovarian cancer, liver cancer andgastric cancer. In some embodiments, the cancer is pancreatic cancer.

Matrix Metalloproteinases (MMPs)

In some embodiments, the cysteamine product inhibits enzymatic activityof one or more matrix metalloproteinases (MMPs) in a cancer cellselected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-4, MMP-5,MMP-6, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 and MMP-14.In some embodiments, the cysteamine product inhibits the enzymaticactivity of MMP-2, MMP-9, MMP-12 and/or MMP-14 in a cancer cell. MMPsare a group of zinc-dependent endopeptidases implicated in mammalianangiogenesis, wound healing, and tissue remodeling (17). In cancer, MMPsplay an important role in cell invasion and metastasis by controllingdegradation of the extracellular matrix (18). In particular, MMP-9 playsa central role in pancreatic cancer invasion, and its inhibitiondecreases liver metastasis of pancreatic cancer (15,19). Hence, many MMPinhibitors of broad to narrow specificity have been investigated fortheir anti-cancer effects. Some MMP inhibitors successfully suppresstumor growth and metastasis in animal models (20-22), but they fail toshow anti-cancer effects in clinical settings.

Cysteamine/Cystamine

Cysteamine plays a role in formation of the protein glutathione (GSH)precursor. In cystinosis, cysteamine acts by converting cystine tocysteine and cysteine-cysteamine mixed disulfide which are then bothable to leave the lysosome through the cysteine and lysine transportersrespectively (Gahl et al., N Engl J Med 2002; 347(2):111-21). Within thecytosol the mixed disulfide can be reduced by its reaction withglutathione and the cysteine released can be used for further GSHsynthesis. The synthesis of GSH from cysteine is catalyzed by twoenzymes, gamma-glutamylcysteine synthetase and GSH synthetase. Thispathway occurs in almost all cell types, with the liver being the majorproducer and exporter of GSH. The reduced cysteine-cysteamine mixeddisulfide will also release cysteamine, which, in theory is then able tore-enter the lysosome, bind more cystine and repeat the process (Dohilet al., J Pediatr 2006; 148(6):764-9). In a recent study in childrenwith cystinosis, enteral administration of cysteamine resulted inincreased plasma cysteamine levels, which subsequently caused prolongedefficacy in the lowering of leukocyte cystine levels (Dohil et al., JPediatr 2006; 148(6):764-9). This may have been due to “re-cycling” ofcysteamine when adequate amounts of drug reached the lysosome. Ifcysteamine acts in this fashion, then GSH production may also besignificantly enhanced.

Cysteamine is a potent gastric acid-secretagogue that has been used inlaboratory animals to induce duodenal ulceration; studies in humans andanimals have shown that cysteamine-induced gastric acid hypersecretionis most likely mediated through hypergastrinemia. Cysteamine iscurrently FDA approved for use in the treatment of cystinosis, anintra-lysosomal cystine storage disorder. In previous studies performedin children with cystinosis who suffered regular upper gastrointestinalsymptoms, a single oral dose of cysteamine (11-23 mg/kg) was shown tocause hypergastrinemia and a 2 to 3-fold rise in gastricacid-hypersecretion, and a 50% rise in serum gastrin levels. Symptomssuffered by these individuals included abdominal pain, heartburn,nausea, vomiting, and anorexia. U.S. patent application Ser. No.11/990,869 and published International Publication No. WO 2007/089670(each of which is incorporated by reference herein in its entirety)showed that cysteamine induced hypergastrinemia arises, in part, as alocal effect on the gastric antral-predominant G-cells in susceptibleindividuals. The data also suggest that this is also a systemic effectof gastrin release by cysteamine. Depending on the route ofadministration, plasma gastrin levels usually peak after intragastricdelivery within 30 minutes whereas the plasma cysteamine levels peaklater.

Subjects with cystinosis are required to ingest oral cysteamine(CYSTAGON®) every 6 hours day and night. When taken regularly,cysteamine can deplete intracellular cystine by up to 90% (as measuredin circulating white blood cells), and this had been shown to reduce therate of progression to kidney failure/transplantation and also toobviate the need for thyroid replacement therapy. Because of thedifficulty in taking CYSTAGON®, reducing the required dosing improvesthe adherence to therapeutic regimen. International Publication No. WO2007/089670 demonstrates that delivery of cysteamine to the smallintestine reduces gastric distress and ulceration and increases AUC.Delivery of cysteamine into the small intestine is useful due toimproved absorption rates from the small intestine, and/or lesscysteamine undergoing hepatic first pass elimination when absorbedthrough the small intestine. A decrease in leukocyte cystine wasobserved within an hour of treatment.

In addition, sulfhydryl (SH) compounds such as cysteamine, cystamine,and glutathione are active intracellular antioxidants. Cysteamineprotects animals against bone marrow and gastrointestinal radiationsyndromes. The rationale for the important anti-oxidant properties of SHcompounds is further supported by observations in mitotic cells. Theseare the most sensitive to radiation injury in terms of cell reproductivedeath and are noted to have the lowest level of SH compounds.Conversely, S-phase cells, which are the most resistant to radiationinjury using the same criteria, have demonstrated the highest levels ofinherent SH compounds. In addition, when mitotic cells were treated withcysteamine, they became very resistant to radiation. It has also beennoted that cysteamine may directly protect cells against inducedmutations. The protection is thought to result from scavenging of freeradicals, either directly or via release of protein-bound GSH. An enzymethat liberates cysteamine from coenzyme A has been reported in avianliver and hog kidney. Recently, studies have reported a protectiveeffect of cysteamine against the hepatotoxic agents acetaminophen,bromobenzene, and phalloidine.

Cystamine, in addition to its role as a radioprotectant, has been foundto alleviate tremors and prolong life in mice with the gene mutation forHuntington's disease (HD). The drug may work by increasing the activityof proteins that protect nerve cells, or neurons, from degeneration.Cystamine appears to inactivate an enzyme called transglutaminase andthus results in a reduction of huntingtin protein (Nature Medicine(2002) 8, 143-149). In addition, cystamine was found to increase thelevels of certain neuroprotective proteins. However, due to the currentmethods and formulation of delivery of cystamine, degradation and pooruptake require excessive dosing.

Cysteamine Products

In another aspect, the disclosure provides cysteamine products for usein the methods described herein.

A “cysteamine product” in the present disclosure refers generally tocysteamine, cystamine, or a biologically active metabolite or derivativethereof, or combination of cysteamine and cystamine, and includescysteamine or cystamine salts, esters, amides, alkylate compounds,prodrugs, analogs, phosphorylated compounds, sulfated compounds,nitrosylated and glycosylated compounds or other chemically modifiedforms thereof (e.g., chemically modified forms prepared by labeling withradionucleotides or enzymes and chemically modified forms prepared byattachment of polymers such as polyethylene glycol). Thus, thecysteamine or cystamine can be administered in the form of apharmacologically acceptable salt, ester, amide, prodrug or analog or asa combination thereof. In various embodiments, the cysteamine productincludes cysteamine, cystamine or derivatives thereof. In any of theembodiments described herein, a cysteamine product may optionallyexclude N-acetylcysteine.

Salts, esters, amides, prodrugs and analogs of the active agents may beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by J. March,“Advanced Organic Chemistry: Reactions, Mechanisms and Structure,” 4thEd. (New York: Wiley-Interscience, 1992). For example, basic additionsalts are prepared from the neutral drug using conventional means,involving reaction of one or more of the active agent's free hydroxylgroups with a suitable base. Generally, the neutral form of the drug isdissolved in a polar organic solvent such as methanol or ethanol and thebase is added thereto. The resulting salt either precipitates or may bebrought out of solution by addition of a less polar solvent. Suitablebases for forming basic addition salts include, but are not limited to,inorganic bases such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimethylamine, or the like. Preparationof esters involves functionalization of hydroxyl groups which may bepresent within the molecular structure of the drug. The esters aretypically acyl-substituted derivatives of free alcohol groups, i.e.,moieties which are derived from carboxylic acids of the formula R—COOHwhere R is alkyl, and typically is lower alkyl. Esters can bereconverted to the free acids, if desired, by using conventionalhydrogenolysis or hydrolysis procedures. Preparation of amides andprodrugs can be carried out in an analogous manner. Other derivativesand analogs of the active agents may be prepared using standardtechniques known to those skilled in the art of synthetic organicchemistry, or may be deduced by reference to the pertinent literature.

In various embodiments, the cysteamine product does not refer tonanoparticles (including, but not limited to, gold, silver, cadmium andiron nanoparticles) comprising cysteamine (e.g., Wu et al.,Nanomedicine: Nanotechnology, Biology and Medicine, 8:860,869, 2011;Ghosh et al., Biomaterials, 34:807-816, 2013; Unak et al., Surf. N.Niointerfaces, 90:217-226, 2012; Petkova et al, m Nanoscale Res. Lett.,7:287, 2012; and U.S. Patent Publication No. 2010/0034735 or cysteamineincorporated into another active agent (e.g., Fridkin et al., J. Comb.Chem., 7:977-986, 2005).

Pharmaceutical Formulations

The disclosure provides cysteamine products useful in the treatment ofcancer (e.g., to inhibit or suppress metastasis of a tumor or thetreatment of pancreatic cancer). To administer cysteamine products topatients or test animals, it is preferable to formulate the cysteamineproducts in a composition comprising one or more pharmaceuticallyacceptable carriers. Pharmaceutically or pharmacologically acceptablecarriers or vehicles refer to molecular entities and compositions thatdo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below, or are approvedby the U.S. Food and Drug Administration or a counterpart foreignregulatory authority as an acceptable additive to orally or parenterallyadministered pharmaceuticals. Pharmaceutically acceptable carriersinclude any and all clinically useful solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like.

Pharmaceutical carriers include pharmaceutically acceptable salts,particularly where a basic or acidic group is present in a compound. Forexample, when an acidic substituent, such as —COOH, is present, theammonium, sodium, potassium, calcium and the like salts, arecontemplated for administration. Additionally, where an acid group ispresent, pharmaceutically acceptable esters of the compound (e.g.,methyl, tert-butyl, pivaloyloxymethyl, succinyl, and the like) arecontemplated as preferred forms of the compounds, such esters beingknown in the art for modifying solubility and/or hydrolysischaracteristics for use as sustained release or prodrug formulations.

When a basic group (such as amino or a basic heteroaryl radical, such aspyridyl) is present, then an acidic salt, such as hydrochloride,hydrobromide, acetate, maleate, pamoate, phosphate, methanesulfonate,p-toluenesulfonate, and the like, is contemplated as a form foradministration.

In addition, compounds may form solvates with water or common organicsolvents. Such solvates are contemplated as well.

The cysteamine products may be administered orally, parenterally,transocularly, intranasally, transdermally, transmucosally, byinhalation spray, vaginally, rectally, or by intracranial injection. Theterm parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intracisternal injection, or infusiontechniques. Administration by intravenous, intradermal, intramusclar,intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonaryinjection and or surgical implantation at a particular site iscontemplated as well. Generally, compositions for administration by anyof the above methods are essentially free of pyrogens, as well as otherimpurities that could be harmful to the recipient. Further, compositionsfor administration parenterally are sterile.

Pharmaceutical compositions of the disclosure containing a cysteamineproduct as an active ingredient may contain pharmaceutically acceptablecarriers or additives depending on the route of administration. Examplesof such carriers or additives include water, a pharmaceuticallyacceptable organic solvent, collagen, polyvinyl alcohol,polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulosesodium, polyacrylic sodium, sodium alginate, water-soluble dextran,carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose,xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin,propylene glycol, polyethylene glycol, Vaseline, paraffin, stearylalcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol,lactose, a pharmaceutically acceptable surfactant and the like.Additives used are chosen from, but not limited to, the above orcombinations thereof, as appropriate, depending on the dosage form ofthe disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the cysteamine product to beadministered can be prepared in a physiologically acceptable vehicle orcarrier. For solutions or emulsions, suitable carriers include, forexample, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers.

A variety of aqueous carriers, e.g., water, buffered water, 0.4% saline,0.3% glycine, or aqueous suspensions may contain the active compound inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

In some embodiments, the cysteamine product disclosed herein can belyophilized for storage and reconstituted in a suitable carrier prior touse. Any suitable lyophilization and reconstitution techniques can beemployed. It is appreciated by those skilled in the art thatlyophilization and reconstitution can lead to varying degrees ofactivity loss and that use levels may have to be adjusted to compensate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

In one embodiment, the disclosure provides use of an enterically coatedcysteamine product composition. Enteric coatings prolong release untilthe cysteamine product reaches the intestinal tract, typically the smallintestine. Because of the enteric coatings, delivery to the smallintestine is improved thereby improving uptake of the active ingredientwhile reducing gastric side effects. Exemplary enterically coatedcysteamine products are described in International Publication No. WO2007/089670.

In some embodiments, the coating material is selected such that thetherapeutically active agent is released when the dosage form reachesthe small intestine or a region in which the pH is greater than pH 4.5.The coating may be a pH-sensitive materials, which remain intact in thelower pH environs of the stomach, but which disintegrate or dissolve atthe pH commonly found in the small intestine of the patient. Forexample, the enteric coating material begins to dissolve in an aqueoussolution at pH between about 4.5 to about 5.5. For example, pH-sensitivematerials will not undergo significant dissolution until the dosage formhas emptied from the stomach. The pH of the small intestine graduallyincreases from about 4.5 to about 6.5 in the duodenal bulb to about 7.2in the distal portions of the small intestine. In order to providepredictable dissolution corresponding to the small intestine transittime of about 3 hours (e.g., 2-3 hours) and permit reproducible releasetherein, the coating should begin to dissolve at the pH range within thesmall intestine. Therefore, the amount of enteric polymer coating shouldbe sufficient to substantially dissolved during the approximate threehour transit time within the small intestine, such as the proximal andmid-intestine.

Enteric coatings have been used for many years to arrest the release ofthe drug from orally ingestible dosage forms. Depending upon thecomposition and/or thickness, the enteric coatings are resistant tostomach acid for required periods of time before they begin todisintegrate and permit release of the drug in the lower stomach orupper part of the small intestines. Examples of some enteric coatingsare disclosed in U.S. Pat. No. 5,225,202 which is incorporated byreference fully herein. As set forth in U.S. Pat. No. 5,225,202, someexamples of coating previously employed are beeswax and glycerylmonostearate; beeswax, shellac and cellulose; and cetyl alcohol, masticand shellac, as well as shellac and stearic acid (U.S. Pat. No.2,809,918); polyvinyl acetate and ethyl cellulose (U.S. Pat. No.3,835,221); and neutral copolymer of polymethacrylic acid esters(Eudragit L30D) (F. W. Goodhart et al., Pharm. Tech., pp. 64-71, April1984); copolymers of methacrylic acid and methacrylic acid methylester(Eudragits), or a neutral copolymer of polymethacrylic acid esterscontaining metallic stearates (Mehta et al., U.S. Pat. Nos. 4,728,512and 4,794,001). Such coatings comprise mixtures of fats and fatty acids,shellac and shellac derivatives and the cellulose acid phthlates, e.g.,those having a free carboxyl content. See, Remington's at page 1590, andZeitova et al. (U.S. Pat. No. 4,432,966), for descriptions of suitableenteric coating compositions. Accordingly, increased adsorption in thesmall intestine due to enteric coatings of cysteamine productcompositions can result in improved efficacy.

Generally, the enteric coating comprises a polymeric material thatprevents cysteamine product release in the low pH environment of thestomach but that ionizes at a slightly higher pH, typically a pH of 4 or5, and thus dissolves sufficiently in the small intestines to graduallyrelease the active agent therein. Accordingly, among the most effectiveenteric coating materials are polyacids having a pKa in the range ofabout 3 to 5. Suitable enteric coating materials include, but are notlimited to, polymerized gelatin, shellac, methacrylic acid copolymertype CNF, cellulose butyrate phthalate, cellulose hydrogen phthalate,cellulose proprionate phthalate, polyvinyl acetate phthalate (PVAP),cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT),hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate, dioxypropyl methylcellulose succinate, carboxymethylethylcellulose (CMEC), hydroxypropyl methylcellulose acetate succinate(HPMCAS), and acrylic acid polymers and copolymers, typically formedfrom methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethylmethacrylate with copolymers of acrylic and methacrylic acid esters(Eudragit NE, Eudragit RL, Eudragit RS). In one embodiment, thecysteamine product composition is administered in an oral deliveryvehicle, including but not limited to, tablet or capsule form. Tabletsare manufactured by first enterically coating the cysteamine product. Amethod for forming tablets herein is by direct compression of thepowders containing the enterically coated cysteamine product, optionallyin combination with diluents, binders, lubricants, disintegrants,colorants, stabilizers or the like. As an alternative to directcompression, compressed tablets can be prepared using wet-granulation ordry-granulation processes. Tablets may also be molded rather thancompressed, starting with a moist material containing a suitablewater-soluble lubricant.

The preparation of delayed, controlled or sustained/extended releaseforms of pharmaceutical compositions with the desired pharmacokineticcharacteristics is known in the art and can be accomplished by a varietyof methods. For example, oral controlled delivery systems includedissolution-controlled release (e.g., encapsulation dissolution controlor matrix dissolution control), diffusion-controlled release (reservoirdevices or matrix devices), ion exchange resins, osmotic controlledrelease or gastroretentive systems. Dissolution controlled release canbe obtained, e.g., by slowing the dissolution rate of a drug in thegastrointestinal tract, incorporating the drug in an in soluble polymer,and coating drug particles or granules with polymeric materials ofvarying thickness. Diffusion controlled release can be obtained, e.g.,by controlling diffusion through a polymeric membrane or a polymericmatrix. Osmotically controlled release can be obtained, e.g., bycontrolling solvent influx across a semipermeable membrane, which inturn carries the drug outside through a laser-drilled orifice. Theosmotic and hydrostatic pressure differences on either side of themembrane govern fluid transport. Prolonged gastric retention may beachieved by, e.g., altering density of the formulations, bioadhesion tothe stomach lining, or increasing floating time in the stomach. Forfurther detail, see the Handbook of Pharmaceutical Controlled ReleaseTechnology, Wise, ed., Marcel Dekker, Inc., New York, N.Y. (2000),incorporated by reference herein in its entirety, e.g. Chapter 22 (“AnOverview of Controlled Release Systems”).

The concentration of cysteamine product in these formulations can varywidely, for example from less than about 0.5%, usually at or at leastabout 1% to as much as 15 or 20% by weight and are selected primarilybased on fluid volumes, manufacturing characteristics, viscosities,etc., in accordance with the particular mode of administration selected.Actual methods for preparing administrable compositions are known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers include,for example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544, 1993).

The enterically coated cysteamine product can comprise variousexcipients, as is well known in the pharmaceutical art, provided suchexcipients do not exhibit a destabilizing effect on any components inthe composition. Thus, excipients such as binders, bulking agents,diluents, disintegrants, lubricants, fillers, carriers, and the like canbe combined with the cysteamine product. Oral delivery vehiclescontemplated for use herein include tablets, capsules, comprising theproduct. For solid compositions, diluents are typically necessary toincrease the bulk of a tablet or capsule so that a practical size isprovided for compression. Suitable diluents include dicalcium phosphate,calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride,dry starch and powdered sugar. Binders are used to impart cohesivequalities to a oral delivery vehicle formulation, and thus ensure that atablet remains intact after compression. Suitable binder materialsinclude, but are not limited to, starch (including corn starch andpregelatinized starch), gelatin, sugars (including sucrose, glucose,dextrose and lactose), polyethylene glycol, waxes, and natural andsynthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, hydroxyethyl cellulose, hypromellose,and the like), and Veegum. Lubricants are used to facilitate oraldelivery vehicle manufacture; examples of suitable lubricants include,for example, magnesium stearate, calcium stearate, and stearic acid, andare typically present at no more than approximately 1 weight percentrelative to tablet weight. Disintegrants are used to facilitate oraldelivery vehicle, (e.g., a tablet) disintegration or “breakup” afteradministration, and are generally starches, clays, celluloses, algins,gums or crosslinked polymers. If desired, the pharmaceutical compositionto be administered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and the like. Ifdesired, flavoring, coloring and/or sweetening agents may be added aswell. Other optional components for incorporation into an oralformulation herein include, but are not limited to, preservatives,suspending agents, thickening agents, and the like. Fillers include, forexample, insoluble materials such as silicon dioxide, titanium oxide,alumina, talc, kaolin, powdered cellulose, microcrystalline cellulose,and the like, as well as soluble materials such as mannitol, urea,sucrose, lactose, dextrose, sodium chloride, sorbitol, and the like.

A pharmaceutical composition may also comprise a stabilizing agent suchas hydroxypropyl methylcellulose or polyvinylpyrrolidone, as disclosedin U.S. Pat. No. 4,301,146. Other stabilizing agents include, but arenot limited to, cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropyl methylcellulose phthalate, microcrystalline cellulose andcarboxymethylcellulose sodium; and vinyl polymers and copolymers such aspolyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonicacid copolymer, and ethylene-vinyl acetate copolymers. The stabilizingagent is present in an amount effective to provide the desiredstabilizing effect; generally, this means that the ratio of cysteamineproduct to the stabilizing agent is at least about 1:500 w/w, morecommonly about 1:99 w/w.

The tablet, capsule, or other oral delivery system is manufactured byfirst enterically coating the cysteamine product. A method for formingtablets herein is by direct compression of the powders containing theenterically coated cysteamine product, optionally in combination withdiluents, binders, lubricants, disintegrants, colorants, stabilizers orthe like. As an alternative to direct compression, compressed tabletscan be prepared using wet-granulation or dry-granulation processes.Tablets may also be molded rather than compressed, starting with a moistmaterial containing a suitable water-soluble lubricant.

In various embodiments, the enterically coated cysteamine product isgranulated and the granulation is compressed into a tablet or filledinto a capsule. Capsule materials may be either hard or soft, and aretypically sealed, such as with gelatin bands or the like. Tablets andcapsules for oral use will generally include one or more commonly usedexcipients as discussed herein.

In a further embodiment, the cystemine product is formulated as acapsule. In one embodiment, the capsule comprises the cysteamine productand the capsule is then enterically coated. Capsule formulations areprepared using techniques known in the art.

A suitable pH-sensitive polymer is one which will dissolve in intestinalenvironment at a higher pH level (pH greater than 4.5), such as withinthe small intestine and therefore permit release of thepharmacologically active substance in the regions of the small intestineand not in the upper portion of the GI tract, such as the stomach.

For administration of the dosage form, i.e., the tablet or capsulecomprising the enterically coated cysteamine product, a total weight inthe range of approximately 100 mg to 1000 mg is used. The dosage form isorally administered to a subject need thereof.

In addition, various prodrugs can be “activated” by use of theenterically coated cysteamine. Prodrugs are pharmacologically inert,they themselves do not work in the body, but once they have beenabsorbed, the prodrug decomposes. The prodrug approach has been usedsuccessfully in a number of therapeutic areas including antibiotics,antihistamines and ulcer treatments. The advantage of using prodrugs isthat the active agent is chemically camouflaged and no active agent isreleased until the drug has passed out of the gut and into the cells ofthe body. For example, a number of produgs use S—S bonds. Weak reducingagents, such as cysteamine, reduce these bonds and release the drug.Accordingly, the compositions of the disclosure are useful incombination with pro-drugs for timed release of the drug. In thisaspect, a pro-drug can be administered followed by administration of anenterically coated cysteamine compositions of the disclosure (at adesired time) to activate the pro-drug.

Dosing and Administration

The cysteamine product is administered in a therapeutically effectiveamount; typically, the composition is in unit dosage form. The amount ofcysteamine product administered is, of course, dependent on the age,weight, and general condition of the patient, the severity of thecondition being treated, and the judgment of the prescribing-physician.Suitable therapeutic amounts will be known to those skilled in the artand/or are described in the pertinent reference texts and literature.Current non-enterically coated doses are about 1.35 g/m² body surfacearea and are administered 4-5 times per day (Levtchenko et al., PediatrNephrol. 21:110-113, 2006). In one aspect, the dose is administeredeither one time per day or multiple times per day. The cysteamineproduct may be administered less than four time per day, e.g., one, twoor three times per day. In some embodiments, an effective dosage ofcysteamine product may be within the range of 0.01 mg to 1000 mg per kg(mg/kg) of body weight per day. In some embodiments, the cysteamine,cystamine or pharmaceutically acceptable salt thereof is administered ata daily dose ranging from about 10 mg/kg to about 250 mg/kg, or fromabout 100 mg/kg to about 250 mg/kg, or from about 60 mg/kg to about 100mg/kg or from about 50 mg/kg to about 90 mg/kg, or from about 30 mg/kgto about 80 mg/kg, or from about 20 mg/kg to about 60 mg/kg, or fromabout 10 mg/kg to about 50 mg/kg. Further, the effective dose may be 0.5mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450mg/kg, 500 mg/kg, and may increase by 25 mg/kg increments up to 1000mg/kg, or may range between any two of the foregoing values. In someembodiments, the cysteamine product is administered at a total dailydose of from approximately 0.25 g/m² to 4.0 g/m² body surface area,e.g., at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m², or up to about 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7,3.0, 3.25, 3.5 or 3.75 g/m² or may range between any two of theforegoing values. In some embodiments, the cysteamine product may beadministered at a total daily dose of about 0.5-2.0 g/m² body surfacearea, or 1-1.5 g/m² body surface area, or 1-1.95 g/m² body surface area,or 0.5-1 g/m² body surface area, or about 0.7-0.8 g/m² body surfacearea, or about 1.35 g/m² body surface area, or about 1.3 to about 1.95grams/m2/day, or about 0.5 to about 1.5 grams/m2/day, or about 0.5 toabout 1.0 grams/m2/day, preferably at a frequency of fewer than fourtimes per day, e.g. three, two or one times per day. Salts or esters ofthe same active ingredient may vary in molecular weight depending on thetype and weight of the salt or ester moiety. For administration ofenteric dosage form, e.g., a tablet or capsule or other oral dosage formcomprising the enterically coated cysteamine product, a total weight inthe range of approximately 100 mg to 1000 mg is used.

Administration may continue for at least 3 months, 6 months, 9 months, 1year, 2 years, or more.

Combination Therapy

Therapeutic compositions can be administered in therapeuticallyeffective dosages alone or in combination with adjunct cancer therapysuch as surgery, chemotherapy, radiotherapy, thermotherapy, and lasertherapy, and may provide a beneficial effect, e.g. reducing tumor size,slowing rate of tumor growth, inhibiting metastasis, or otherwiseimproving overall clinical condition, without necessarily eradicatingthe cancer. Cytostatic and cytotoxic agents that target the cancer cellsare specifically contemplated for combination therapy. Likewise, agentsthat target angiogenesis or lymphangiogenesis are specificallycontemplated for combination therapy.

As used herein, a “chemotherapeutic agent” is a chemical compound usefulin the treatment of cancer. Examples of chemotherapeutic agents include:alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;vinca alkaloids; epipodophyllotoxins; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammall andcalicheamicin omegall; L-asparaginase; anthracenedione substituted urea;methyl hydrazine derivatives; dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, canninomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid:gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitiaerine; pentostatin; phenamet; pirarubicin; losoxantione;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2,2″-trichlorotiiethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine): urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® docetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum coordination complexes such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone;teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluoromethylornithine (DFMO); retinoids such as retinoic acid;capecitabine: leucovorin (LV); irenotecan; adrenocortical suppressant;adrenocorticosteroids; progestins; estrogens; androgens;gonadotropin-releasing hormone analogs; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON-toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASL® exemestane, formestanie, fadrozole, RIVISOR®vorozole, FEMARA® letrozole, and ARTMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF-A expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rJL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELLX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” as used herein refers to a compound orcomposition which inhibits growth of a cell in vitro and/or in vivo.Thus, the growth inhibitory agent may be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL®, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.

Cytokines that are effective in inhibiting tumor metastasis are alsocontemplated for use in the combination therapy. Such cytokines,lymphokines, or other hematopoietic factors include, but are not limitedto, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14. IL-15, IL-16, IL-17, IL-18,IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cellfactor, and erythropoietin.

In some embodiments, the methods described herein further compriseadministering an MMP inhibitor (e.g., a MMP-2 inhibitor, a MMP-12inhibitor and/or a MMP-9 inhibitor) to the subject. As used herein, “MMPinhibitor” is an agent that directly or indirectly inhibits MMPactivity. This includes an agent that blocks MMP activity or an agentthat blocks a pathway of MMP production. The agent causes a reduction inMMP activity in a cancer cell (or cancerous tissue) regardless of themechanism of its action. Representative examples of MMP inhibitorsinclude Tissue Inhibitors of Metalloproteinases (TIMPs) (e.g., TIMP-1,TIMP-2, TIMP-3, or TIMP-4), O2-macroglobulin, tetracyclines (e.g.,tetracycline, minocycline, and doxycycline), hydroxamates (e.g.,BATIMASTAT, MARIMISTAT and TROCADE), chelators (e.g., EDTA, cysteine,acetylcysteine, D-penicillamine, and gold salts), synthetic MMPfragments, succinyl mercaptopurines, phosphonamidates, and hydroxaminicacids.

Broad-spectrum inhibitors that inhibit more than one type of MMP arealso contemplated. Exemplary broad spectrum MMP inhibitors include, butare not limited to, GM6001, batimastat, marimastat, prinomastat, BAY12-9566, MM1270(B), BMS-275291, and metastat. Inhibitors that arecapable of inhibiting MMP2, MMP9 or both MMP2 and MMP9 are specificallycontemplated. An exemplary MMP-2/MMP-9 inhibitor includes, but is notlimited to, SB-3CT. For example, in one embodiment, combination therapycomprising the administration of the cysteamine product and aMMP-2/MMP-9 inhibitor is specifically contemplated.

Assays for measuring MMP inhibition/suppression are readily known in theart, and include, for example, the following: Cawston T. E., Barrett A.J., “A rapid and reproducible assay for collagenase using (14C)acetylated collagen,” Anal. Biochem. 35:1961-1965 (1963); Cawston T. E.,Murphy G. “Mammalian collagenases,” Methods in Enzymology 80:711 (1981);Koshy P. T. J., Rowan A. D., Life P. F., Cawston T. E., “96-well plateassays for measuring collagenase activity using (3)H-acetylatedcollagen,” Anal. Biochem. 99:340-345 (1979); Stack M. S., Gray R. D.,“Comparison of vertebrate collagenase and gelatinase using a newfluorogenic substrate peptide,” J. Biol. Chem. 264:4277-4281 (1989); andKnight C. G, Willenbrock F., Murphy G, “A novel coumarin-labelledpeptide for sensitive continuous assays of the matrixmetalloproteinases,” FEBS Lett 296:263-266 (1992).

The treatment methods described herein optionally include monitoring theeffect of the therapeutic composition on the tumor. For example, thesize of the tumor can be determined, as can the presence of metastases.Also contemplated is measurement of the degree of metastasis, e.g., bymeasuring the number of metastatic modules or by measurement of ascitesassociated with metastasis.

The cysteamine product and other drugs/therapies can be administered incombination either simultaneously in a single composition or in separatecompositions. Alternatively, the administration is sequential.Simultaneous administration is achieved by administering a singlecomposition or pharmacological protein formulation that includes boththe cysteamine product and other therapeutic agent(s). Alternatively,the other therapeutic agent(s) are taken separately at about the sametime as a pharmacological formulation (e.g., tablet, injection or drink)of the cysteamine product.

In various alternatives, administration of the cysteamine product canprecede or follow administration of the other therapeutic agent(s) byintervals ranging from minutes to hours. For example, in variousembodiments, it is further contemplated that the agents are administeredin a separate formulation and administered concurrently, withconcurrently referring to agents given within 30 minutes of each other.

In embodiments where the other therapeutic agent(s) and the cysteamineproduct are administered separately, one would generally ensure that thecysteamine product and the other therapeutic agent(s) are administeredwithin an appropriate time of one another so that both the cysteamineproduct and the other therapeutic agent(s) can exert, synergistically oradditively, a beneficial effect on the patient. For example, in variousembodiments the cysteamine product is administered within about 0.5-6hours (before or after) of the other therapeutic agent(s). In variousembodiments, the cysteamine product is administered within about 1 hour(before or after) of the other therapeutic agent(s).

In another aspect, the second agent is administered prior toadministration of the cysteamine composition. Prior administrationrefers to administration of the second agent within the range of oneweek prior to treatment with cysteamine, up to 30 minutes beforeadministration of cysteamine. It is further contemplated that the secondagent is administered subsequent to administration of the cysteaminecomposition. Subsequent administration is meant to describeadministration from 30 minutes after cysteamine treatment up to one weekafter cysteamine administration.

Animal Models

Cysteamine products can be evaluated in animal models known in the artfor the disease indications contemplated herein. Exemplary animal modelsfor metastatic cancers are described in Kim et al., (Biochem. Biophys.Res. Comm., 394:443-447, 2010) (breast cancer); Bresalier et al., (J.Clin. Invest., 87:1037-1045, 1991) (colorectal cancer); Furukawa et al.,Cancer Res., 53:1204, 1993) (stomach cancer); Gingrich et al., (CancerRes., 56:4096, 1996) (prostate cancer); Cranmer et al., (Melanoma Res.,15:325-356, 2005) (melanoma); Zheng et al., Oncogene, 26:6896-6904,2007) (lung cancer); and Wang et al., (Neuropathol. Appl. Neurobiol.,37:189-205, 2011) (brain cancer).

Kits

The disclosure also provides kits for carrying out the methods of thedisclosure. In various embodiments, the kit contains, e.g., bottles,vials, ampoules, tubes, cartridges and/or syringes that comprise aliquid (e.g., sterile injectable) formulation or a solid (e.g.,lyophilized) formulation. The kits can also contain pharmaceuticallyacceptable vehicles or carriers (e.g., solvents, solutions and/orbuffers) for reconstituting a solid (e.g., lyophilized) formulation intoa solution or suspension for administration (e.g., by injection),including without limitation reconstituting a lyophilized formulation ina syringe for injection or for diluting concentrate to a lowerconcentration. Furthermore, extemporaneous injection solutions andsuspensions can be prepared from, e.g., sterile powder, granules, ortablets comprising a cysteamine product-containing composition. The kitscan also include dispensing devices, such as aerosol or injectiondispensing devices, pen injectors, autoinjectors, needleless injectors,syringes, and/or needles. In various embodiments, the kit also providesan oral dosage form, e.g., a tablet or capsule or other oral formulationdescribed herein, of the cysteamine product for use in the method. Thekit also provides instructions for use.

While the disclosure has been described in conjunction with specificembodiments thereof, the foregoing description as well as the exampleswhich follow are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art.

EXAMPLES Materials and Methods

Cell Culture and Reagents:

Pancreatic cancer cell lines (HS766T, MIA-PaCa2, Pane-1, ASPC-1, PK-1,Mpanc96, BxPC-3, KLM, HPAF-II, and SW1990) were obtained from theAmerican Type Culture Collection (Manassas, Va.). Cysteaminehydrochloride was purchased from Sigma-Aldrich (St. Louis, Mo.) anddissolved in distilled water.

Cell Migration Assay:

Cell migration assay is a classical wound healing assay (25). For thisassay, cells were cultured in 10 cm petri dishes until they werecompletely confluent. Cell monolayers were scraped with a sterile yellowmicropipette tip and washed with PBS three times; the cells were thencultured in medium containing 0-5 mM of cysteamine for 24 hours. Fiverandom fields were selected and pictures were taken using an invertedmicroscope. The average area (mm 2) of the gap between the cell layerswas calculated using IPLab imaging software (BD Biosciences-Bioimaging,Rockville, Md.).

Matrigel Invasion Assay.

Cell invasion was assayed in BD BioCoat Matrigel invasion chambers (BDBiosciences; 24 wells, 8 p.m pore size) as described previously (26).Briefly, cells were incubated with different concentrations ofcysteamine (0-5 mM) for 24 hours. Non-invaded cells were removed fromthe upper surface of the membrane with a cotton swab, and cells on thelower surface of the membrane were fixed and stained with H&E. Threerandom fields per chamber were counted. Data were shown as mean±S.D. oftriplicate determinations.

Cell Viability Assay:

Cell viability of pancreatic cancer cell lines was measured by countingviable cell numbers. Briefly, 3×10⁵ cells were seeded per well in a6-well plate with 2.0 milliliter complete medium and incubated overnightfor plating. The cells were treated with different concentrations ofcysteamine for 24-48 hours. After incubation, cells were detached withtrypsin, washed and stained with 0.4% trypan blue. Cell number wasmanually counted using a hemocytometer and presented as number of viablecells per milliliter.

Measurement of MMP Activity:

MMPs activity was measured by Mca-KPLGL-Dpa-AR-NH2 Fluorogenic PeptideSubstrate (R&D systems, Emeryville, Calif.). Total cell protein fromeach pancreatic cancer cell line was collected using lysis buffer(containing 50 mM Tris-HCl, 10 mM CaCl₂, 0.05% Brij35 and 0.25%Triton-X) without an MMP inhibitor. Substrate and 10 μg of total cellprotein in the buffer were mixed in a black-wall 96-well plate. After 1hour incubation, fluorescence units were determined usingexcitation/emission=320/405 nm.

Substrate Gel Zymographic Assay for MMP Activity:

Gelatin gel zyrnography was performed on cell lysates from H57666 andMIA-PaCa2 pancreatic cancer cell line with or without culture withdifferent concentration of cysteamine essentially as previouslydescribed (27). Briefly, 50 μg of total protein was electrophoresed on10% SDS-PAGE containing 0.1% gelatin as substrate. After washing with 1%Triton X-100 in 50 mM Tris/HC 1, pH 7.5 for 1 hr to remove SDS, the gelswere incubated overnight at 37° C. in 50 mM Tris/HCl, pH 7.5, containing150 mM NaCl, 10 mM CaCl₂ and 0.1% Triton X-100, prior to staining withsimply blue safe stain. The gels were washed with deionized water atroom temperature. Gelatin-degrading enzymes were identified by theirability to digest gelatin as demonstrated by clear zones of digestedgelatin. Relevant band intensities were quantified by scanningdensitometric analysis and normalized to cell number.

Quantitative Reverse Transcription-PCR (qRT-PCR):

qRT-PCR was performed as described previously using QuantiTect SYBRGreen PCR Kits (QIAGEN, Valencia, Calif.) (28). Gene specific primersfor human MMP-9, MMP-12, MMP-14 and 13-actin were either purchased fromQIAGEN or synthesized at the CBER core facility. Gene expression wasnormalized to β-actin before the fold change in gene expression wasdetermined.

Enzyme-Linked ImmunoSorbent Assay (ELISA):

The NIMP-9 protein level was determined using Human total MMP-9 DuoSetkit (R&D systems, Emeryville, Calif.) following the manufacturer'sinstruction. Ten microgram of total protein was aliquoted in each welland the MMP-9 concentration was determined by the colorimetric method.

IC₅₀ of Cysteamine Against MMPs Activity:

IC₅₀ of cysteamine and batimastat was determined using a fluorimetricMMP inhibitor profiling kit (Enzo Life Science, Farmingdale, N.Y.),following the manufacturer's instruction. Briefly, each MMP enzyme wasmixed with various concentrations of cysteamine and batimastat in ablack-wall 96 well plate and incubated for 30 minutes. Afteradministration of fluorogenic substrate, a velocity of fluorescentincrease was measured using excitation/emission=320/405 nm.

Measurement of Metastasis, Survival and Body Weight of Animals Implantedwith Orthotopic Pancreatic Cancer in Mouse Model:

Female nude nu/nu mice between ages 5 and 6 weeks were maintained in abarrier facility in HEPA-filtered rack. All animal studies wereconducted under approved protocol #2000-06 by the CBER InstitutionalAnimal Care and Use Committee in accordance with the principles andprocedures outlined in the NIH Guideline for the Care and Use ofLaboratory Animals. For orthotopic tumor cell injection, the pancreaswas carefully exposed, and 2.0×10⁶ of MIA-PaCa2 and H5766T cells wereinjected into the organ. The pancreas was then returned to theperitoneal cavity, and the abdominal wall and skin were closed with skinclips (29, 30). From day 4 after tumor implantation, increasing doses ofcysteamine (0, 25, 100, or 250 mg/kg/day) were subcutaneously injectedtwice a day until the end of the experiment. At day 30, mice weresacrificed and the number of metastatic nodules visible by naked eye andwhose sizes were >5 mm in diameter counted. The total weight of primarytumor and metastatic nodules were also measured. Pictures were takenimmediately after sacrificing the animals. Additionally, mouse survivaltime was monitored in an independent experiment. Mice were sacrificedwhen they had severe ascites or cachexia. Mice in both tumor models wereweighed at day 10 and day 25 after Cysteamine treatment.

Measurement of Enzymes in Mouse Serum:

Mouse blood was collected from the tail vein. Serum levels of alanineaminotransferase (ALT), creatine kinase (CK), aldolase and creatinine(Cr) were measured using different kits obtained from Pointe Scientific,Inc. (Canton, Mich.) and Caldon Biotech, Inc. (Vista, Ca), following themanufacturer's instruction.

MMP Activity and MMP-9 Expression in Primary Tumors:

When mice were sacrificed at day 30, primary tumors were collected. Forextraction of total protein, the tumor was soaked with lysis buffer andhomogenized using TissueRuptor (QIAGEN): the supernatant was collected.Total RNA was extracted using FastRNA pro green kit (MP Biomedicals,Solon, Ohio) following the manufacturer's instructions. MMP activity,gelatin hydrolyzing MMP activity and the mRNA and protein levels ofMMP-9 were determined as mentioned above.

Statistical Analysis:

The data for enzymatic activity, ELISA, and qRT-PCR were comparedbetween each group by ANOVA. Survival curves were generated by theKaplan-Meier method and compared by using the log-rank test.

Example 1 Cysteamine Inhibits Pancreatic Cancer Cell Migration andInvasion without being Cytotoxic to Cells

The following example describes the effect of cysteamine on pancreaticcancer cell invasion and migration.

The effect of cysteamine on pancreatic cancer cell invasion wasdetermined using a matrigel invasion assay. For this assay, tendifferent pancreatic cancer cell lines in a matrigel invasion chamberwere used. Results showed that cysteamine inhibited cell invasion in aconcentration dependent manner. Even the lowest concentration ofcysteamine (0.05 mM) significantly inhibited cell invasion in allpancreatic cancer cell lines.

The effect of cysteamine on migration of the same pancreatic cancer celllines in the invasion assay was also determined. For the migrationassay, a wound healing assay was used (described above in Example 1).Results indicated that cell migration was significantly inhibited at andabove 0.05 mM of cysteamine in all ten cell lines. Because cysteaminemediated similar effects in all ten cell lines used in these assays, tworandom cell lines were chosen for all other assays.

The cytotoxicity of cysteamine against two pancreatic cancer cell lines(H5766T and MIA-PaCa2) was also examined and the number of viable cellsafter 24 and 48 hour cysteamine treatment were counted. Cysteamineshowed cell toxicity at >12.5 mM concentration in both cell lines testedbut no evidence of toxicity was observed at lower concentrations as thenumber of viable cells was similar to untreated cells. These resultssuggest that both cell migration and invasion were inhibited at anoncytotoxic concentration of cysteamine.

Example 2 Cysteamine Inhibits MMP Enzymatic Activity in Pancreatic CellLines

The following Example investigated the mechanism of inhibition of cellmigration and invasion by cysteamine, by evaluating the effect ofcysteamine on MMP enzymatic activity.

Cysteamine inhibited MMP activity in two representative pancreaticcancer cell lines tested in a concentration-dependent manner. The IC₅₀(the concentration of cysteamine at which 50% MMP enzyme activity(proteolysis) is inhibited) was calculated by ELISA (the results ofwhich are provided below in Table 1).

TABLE 1 MMP profiling. MMP profiling, IC₅₀ Cysteamine (μM) Batimastat(nM) MMP-1 290 13 MMP-2 38 5.4 MMP-3 330 300 MMP-7 40 29 MMP-8 240 9.8MMP-9 200 6.7 MMP-10 310 52 MMP-12 60 12 MMP-13 460 6 MMP-14 91 130

Cysteamine directly inhibited each MMP enzymatic activity, with an IC₅₀of 38-460 μM. Since MMP-9 plays a central role in pancreatic cancerinvasion, MMP-9 mRNA and protein levels were examined in both celllines. In contrast to the enzymatic activity, protein and mRNA levels ofMMP-9 were modestly increased at the highest concentration of cysteamine(5 mM). mRNAs for two other MMPs (i.e., MMP-12 and MMP-14) showed amodest increase similar to MMP-9 in response to cysteamine treatment. Incontrast, zymographic gelatin hydrolyzing activity results for MMP-9differed significantly from mRNA expression and both pro and activeMMP-9 activities decreased significantly in a dose dependent manner.

Example 3 Cysteamine Decreases Metastasis and Prolongs Survival ofImmunodeficient Mice Implanted Orthopically with Human Pancreatic Cancer

The following example investigated the anti-metastasis effect ofcysteamine in two orthotopic mouse models using human pancreatic cancercell lines.

Mice were treated twice daily with cysteamine subcutaneously from day 4after tumor implantation until the end of the experiment. At day 30, thenumber and weight of the primary tumor and metastatic nodules weremeasured. In both tumor models, the size and weight of the primarytumors did not show any difference between control and treated groups.However, cysteamine significantly decreased the number of metastaticnodules in a dose-dependent manner. At the highest dose (250 mg/kg/day),cysteamine significantly decreased the number of metastases of H5766Ttumor by ˜90% (from 34 to 3.6). The total weight of metastatic nodulesof MIA-PaCa2 tumor was also decreased by ˜90% at the highest dose. Inaddition, two of 5 mice in the H5766T tumor model and 4 of 5 mice inMIA-PaCa2 tumor model developed ascites in their peritoneal cavity.However, no mice developed ascites at 100 and 250 mg/kg/day dose ineither tumor model. The survival of mice among different treatmentgroups was also determined. Mouse survival time was significantlyprolonged when treated with 100 mg/kg/day cysteamine in both tumormodels (FIGS. 1A and 1B).

The general condition and body weight of mice throughout theexperimental period was also monitored. There was no significantdifference in general appearance and body weight among four groups ofanimals in both tumor models. Similarly, there was no alteration inserum enzymes representing liver function (ALT) or muscle damage(Aldolase) nor evidence of skeletal muscle damage or kidney function(creatinine kinase and creatinine) in the cysteamine-treated groups.Moreover, no organ toxicity was detected in any vital organ such as theliver, kidney, brain, heart, and lung in cysteamine-treated mice whenevaluated by histological examination.

Example 4 Cysteamine Decreases MMP Activity in Primary Orthotopic Tumors

MMP activity in primary orthotopic tumors was measured at day 30.Cysteamine decreased MMPs activity in both tumors (H5766T and MIA-PaCa2)at 100 and 250 mg/Kg cysteamine doses (FIG. 8A). In the same samples,mRNA was measured by q-RT-PCR and protein levels of MMP-9 by ELISA. Incontrast to in vitro results, cysteamine did not affect mRNA and proteinlevels of MMP-9 in primary tumors harvested from mice. However,zyrnography assay for MMP-9 showed a dose dependent decrease ingelatinase activity.

DISCUSSION

These results demonstrate that cysteamine inhibits pancreatic cancercell migration and invasion through direct inhibition of MMP enzymaticactivity in vitro. This newly discovered property of cysteamine resultedin inhibition of metastasis of human pancreatic cancer cellsorthotopically implanted onto pancreas of immunodeficient mice; theeffect was cysteamine dose-dependent. Cysteamine decreased not only thenumber of metastasis in the peritoneal cavity, but also decreased theascites generated by aggressive pancreatic tumor metastasis. Incontrast, no significant change in the size or weight of the primarypancreatic tumor was observed. Consistent with this observation,cysteamine did not cause any effect on cell viability in pancreaticcancer lines up to a concentration which caused significant inhibitionof cell migration and invasion. Mice treated with cysteamine survivedlonger compared to control mice treated with excipient. Both in vitroand in primary tumors in vivo, MMP enzymatic activity decreased withcysteamine treatment while their expression at mRNA and protein levelsdid not change Zymographic results confirming that cysteamine inducedMVP inhibition in vitro and in vivo. These observations indicate thatblocking of catalytic activities of MMPs by cysteamine plays a role inthe inhibition of tumor metastasis in animal model of pancreatic cancer.

The anti-metastatic effects of cysteamine were mediated without anyvisible signs of toxicity. Mice treated with even the highest dose ofcysteamine (250 mg/kg/day) displayed no adverse effects related togeneral appearance, body weight, muscle damage, serum enzymes and serumcreatinine levels. In addition, major organs from treated animals showedno evidence of histological damage. These observations are consistentwith the known safety profile of cysteamine in humans (2,3). Cysteaminecaused only gastrointestinal symptoms in subjects, as it increasedgastric acid production and decreased gastrointestinal motility (31).However, these effects were controlled by concomitant use of proton-pumpinhibitor (32). It is noteworthy that cysteamine sufficiently inhibitedcancer cell migration and invasion in vitro at 50 μM, a concentrationthat can be achieved in vivo. Oral administration of cysteamine (givenevery 6 hours at 60 to 90 mg/kg of body weight per day) can increase thecysteamine plasma level up to ˜50 μM (3.33-35). In addition, 100mg/kg/day s.c. injection of cysteamine in animals is similar to thedosage used for cystinosis, and this dose produced a significantdecrease in tumor metastasis and prolongation of survival. It iscontemplated that cysteamine may be safely administered in the clinic tocontrol pancreatic cancer metastasis.

Both mRNA and protein levels for MMP-9 were modestly to moderatelyupregulated in vitro at the highest dose of cysteamine. Similarly, mRNAof MMP-12 and 14 were also modestly upregulated at the highest dose.This increase in MMP-9 was not observed in vivo, perhaps because thepancreatic cancer cells were incubated with cysteamine only for 24hours, while in vivo tumors were exposed to cysteamine continuously for27 days. Cysteamine levels in tumors in vivo were not measured becauseintracellular metabolic fate of cysteamine in vivo is complex anddifficult to measure as it binds to free thiol, particularly thecysteines of cellular proteins. Instead, activity and cysteamine levelswere extrapolated to the biological effects of cysteamine on MMPactivities and tumor growth. Nevertheless, the increased MMP-9, MMP-12and MMP-14 levels at the highest concentration in vitro may represent atemporary compensatory effect of cells due to a sudden decrease of MMPactivity by cysteamine. In contrast, the catalytic activity of MMP-9 tohydrolyze gelatin in zymography assay did not show such upregulationafter treatment with highest dose of cysteamine of pancreatic cancercell lines in vitro as well as orthotopic tumors in vivo. In fact inzymography assay for MMP-9, cysteamine caused a dose dependent decreasein gelatinase activity. These results suggest that enzymatic inhibitionof MMP-9 by cysteamine may be involved in decrease of invasion andmetastasis of pancreatic cancer. MMP1, MMP2, MMP7, and MMP9 have alsobeen shown to be expressed in pancreatic tumor (36,37), but the effectof cysteamine on protein and mRNA levels for all these MMPs was notexamined herein. However, it is important to point out that cysteamineinhibited enzymatic activity of MMPs, which includes all MMPs.

The anti-MMP activity of cysteamine was lower than that of specific MMPinhibitors such as batimastat and marimastat (IC₅₀ nM to low μM comparedto high μM range for cysteamine), but cysteamine may be better toleratedin vivo. In fact, batimastat had problems of poor oral bioavailability(24) and marimastat failed in the clinic as higher dosage produced highmusculoskeletal toxicities and poor survival in patients with breastcancer metastasis (38). In the present study, mice tolerated up to 250mg/kg/day cysteamine without any visible, biochemical or histologicalevidence of toxicity or muscular damage.

In previous cancer treatment studies, cysteamine was used because of itsanti-oxidative and radio-protective effects (39). During these studies,it was observed that cysteamine also had anti-carcinogenic andanti-proliferative activities in a variety of cancers. It is reportedherein and in Fujisawa et al (PlosOne, 7:e34437, 2012) that cysteamineexerts anti-MMP and anti-metastatic effects. However, in this studycysteamine did not affect the primary pancreatic tumor size. Based onthese insights, it is proposed that cysteamine can be useful asmono-therapy prior to surgery to prevent metastasis, as an adjuvant, oras a component of combination therapy for advanced stage disease toprolong survival of patients with pancreatic cancer.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

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What is claimed:
 1. A method of inhibiting or suppressing metastasis ofpancreatic cancer in a human subject comprising administeringcysteamine, cystamine or a pharmaceutically acceptable salt thereof tothe subject in an amount effective to inhibit metastasis of the cancer,but less than an amount cytotoxic to the cancer, and wherein thecysteamine, cystamine or pharmaceutically acceptable salt thereof isadministered at a daily dose of at least 10 mg/kg.
 2. A method ofreducing metastasis of pancreatic cancer in a human subject comprisingadministering cysteamine, cystamine or a pharmaceutically acceptablesalt thereof to the subject in an amount effective to reduce metastasisof the cancer, which amount is less than an amount cytotoxic to thecancer.
 3. The method of claim 1 wherein the cysteamine, cystamine orpharmaceutically acceptable salt thereof is administered orally.
 4. Themethod of claim 1 wherein the cysteamine, cystamine or pharmaceuticallyacceptable salt thereof is formulated for delayed release.
 5. The methodof claim 4, wherein the delayed release formulation comprises an entericcoating that releases the cysteamine, cystamine, or pharmaceuticallyacceptable salt thereof when the formulation reaches the small intestineor a region of the gastrointestinal tract of a subject in which the pHis greater than about pH 4.5.
 6. The method of claim 1 wherein thecysteamine, cystamine or pharmaceutically acceptable salt thereof isadministered less than four times a day.
 7. The method of claim 1wherein the cysteamine, cystamine or pharmaceutically acceptable saltthereof is administered twice a day.
 8. The method of claim 1 whereinthe administering results in increased thiol levels compared to levelsbefore administration of the cysteamine, cystamine or pharmaceuticallyacceptable salt thereof.
 9. The method of claim 1 wherein thecysteamine, cystamine or pharmaceutically acceptable salt thereof isformulated in a tablet or capsule which is enterically coated.
 10. Themethod of claim 1 further comprising administering to the subjectadjunct cancer therapy.
 11. The method of claim 10, wherein the adjunctcancer therapy is selected from the group consisting of chemotherapy,surgery, radiotherapy, thermotherapy, cancer vaccination, immunotherapy,gene therapy and laser therapy.
 12. The method of claim 1 furthercomprising administering a further therapeutic agent selected from thegroup consisting of an MMP inhibitor, a chemotherapeutic agent, a growthinhibitory agent, a cancer vaccine, a gene therapy product, animmunotherapy and a cytokine.
 13. The method of claim 1 wherein thecysteamine modulates enzymatic activity of a matrix metalloproteinase(MMP).
 14. The method of claim 13, wherein the enzymatic activity of theMMP is decreased in a primary tumor.
 15. The method of claim 1 whereinthe cysteamine, cystamine or pharmaceutically acceptable salt thereofdecreases metastatic nodules in the subject.
 16. The method of claim 1wherein the cysteamine, cystamine or pharmaceutically acceptable saltthereof decreases ascites in the subject.
 17. The method of claim 1wherein the cysteamine, cystamine or pharmaceutically acceptable saltthereof is administered in a dose up to about 250 mg/kg.
 18. A method ofdecreasing matrix metalloproteinase (MMP) enzymatic activity in a humanpancreatic cancer cell comprising contacting the cancer cell withcysteamine, cystamine or a pharmaceutically acceptable salt thereof inan amount effective to decrease MMP enzymatic activity in the cancercell.
 19. The method of claim 18 wherein the MMP is selected from thegroup consisting of MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7,MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13 and MMP-14.
 20. A method ofreducing metastasis in a human patient afflicted with pancreatic cancercomprising administering to the patient an amount of cysteamine,cystamine, or pharmaceutically acceptable salt thereof in an amounteffective to reduce metastasis of the cancer, the amount being less thanan amount cytotoxic to the cancer, to reduce metastasis of the cancer.21. The method of claim 20, wherein the administration is prior tosurgery, to prevent metastasis.
 22. A method of prolonging survival in ahuman patient afflicted with pancreatic cancer comprising administeringto the patient an amount of cysteamine, cystamine, or pharmaceuticallyacceptable salt thereof in an amount effective to prolong survival ofthe patient, the amount being less than an amount cytotoxic to thecancer.
 23. The method of claim 1, wherein the cysteamine, cystamine orpharmaceutically acceptable salt thereof is conjugated withnanoparticles.